The present invention relates to a novel antibiotic produced by streptococcus mutans, salts thereof, pharmaceutical compositions containing same, and to methods for the production and extraction thereof.
Bacteriocins produced by Gram-positive bacteria can be defined as inhibitory substances having a bactericidal mode of action and an essential protein moiety (Jack et al., 1995, Microbiol. Rev. 59:171-200). Bacteriocins produced by Streptococcus mutans were termed mutacins by Hamada and Ooshima (Hamada et al, 1975, Arch. Oral Biol. 20:641-648). Although there are many reports which show that S. mutans produces inhibitory substances, only a few such inhibitors have been isolated and characterized as mutacins (Chikindas et al., 1995, Antimicrob. Agents Chemother. 39:2656-2660; Delisle, 1986, Microbios 46:21-28; Fukushima et al., 1985, Arch. Oral Biol. 30:229-234; Hamada et al., 1986, Zentralbl. Bakteriol. Hyg. A. 261:287-298; Hillman et al., 1984, Infect. Immun. 44:141-144; Kurita et al., 1988, J. Gen. Microbiol. 134:213-220; Loyola-Rodriguez et al., 1992, J. Gen. Microbiol. 138:269-274; Novak et al., 1994, J. Bacteriol. 176:4316-4320; Novxc3xa1k et al., 1996, Anal. Biochem. 236:358-360; Paul et al., 1975, Infect. Immun. 12:1375-1385; Takada et al., 1984, Infect. Immun. 44:370-378). The well studied mutacins include: RM-10 (Fukushima et al., 1985, Arch. Oral Biol. 30:229-234), C3603 (Takada et al., 1984, Infect. Immun. 44:370-378), JH 1000 (Hillman et al., 1984, Infect. Immun. 44:141-144), GS-5 (Paul et al., 1975, Infect. Immun. 12:1375-1385), MT 3791 and MT 6223 (Hamada et al., 1986, Zentralbl. Bakteriol. Hyg. A. 261:287-298;Loyola-Rodriguez et al., 1992, J. Gen. Microbiol. 138:269-274), mutacin-b (Delisle, 1986, Microbios 46:21-28), mutalipocins (Kurita et al., 1988, J. Gen. Microbiol. 134:213-220) and J-T8 (Chikindas et al., 1995, Antimicrob. Agents Chemother. 39:2656-2660; Novxc3xa1k et al., 1994, J. Bacteriol. 176:4316-4320;Novxc3xa1k et al., 1996, Anal. Biochem. 236:358-360).
A preliminary classification of mutacins has been published previously (Morency et al., 1995, Can. J. Microbiol. 41:826-831), but a definitive classification will have to await the complete chemical characterization of these substances. Among the known mutacins, only T8, which belongs to group J (Morency et al., 1995, Can. J. Microbiol. 41:826-831), was identified as a lantibiotic and its first 8 N-terminal amino acid residues were determined by Novxc3xa1k et al. (Novxc3xa1k et al., 1994, J. Bacteriol. 176:4316-4320). Lantibiotics are defined as bacterium-derived ribosomally synthesized lanthionine-containing peptides with antibiotic activity (Jack et al., 1995, Microbiol. Rev. 59:171-200; Bierbaum et al., 1993, Zentralbl. Bakteriol. 278:1-22;Jack et al., 1995, Trends Biotechnol. 13:269-278). They generally contain unsaturated amino acids like 2,3-didehydroalanine (dhA or U) (2)-2,3-didehydrobutyrine (dhB or O), and 2-aminobutyric acid (Abu). The lantibiotics are divided into two types (Jack et al., 1995, Microbiol. Rev. 59:171-200; Bierbaum et al., 1993, Zentralbl. Bakteriol. 278:1-22; Jack et al., 1995, Trends Biotechnol. 13:269-278; Jung, 1991, in: Nisin and Novel Lantibiotics., Jung et al., eds., pp. 1-34. ESCOM Science, Leiden). Type A comprises screw-shaped, amphipathic molecules with molecular masses between 2151 and 4635 Da and with 2 to 7 net positive charges. Type B consists of more globular molecules with molecular masses between 1825 and 2042 Da and with either no net charge or a net negative charge. They usually contain a higher proportion of modified amino acid residues than type A.
Only four streptococcal strains were shown to produce a lantibiotic: Streptococcus pyogenes producing streptococcin A-FF22 (Jack et al., 1994, Eur J Biochem 220:455-462), Streptococcus salivarius producing salivaricin A (Ross et al., 1993, Appl. Environ. Microbiol. 59:2014-2021), Streptococcus mutans T8 producing mutacin J-T8 (Parrot et al., 1990, Can. J. Microbiol. 36:123-130; Parrot et al., 1992, Congrxc3xa8s de I""AMQ, p. 22; Novak et al., 1993, ASM meeting Abstr. No. A-44, p. 8; Novak et al., 1994, J. Bacteriol 196:4316-4320).
Streptococcus mutans strain B-Ny266 has been previously shown to inhibit 98% of the S. mutans strains tested (Morency et al., 1995, Can. J. Microbiol. 41:826-831) and all tested Gram-positive bacteria including Listeria monocyto genes, Clostridium sporogenes, Mycobacterium phlexc3xaf, Staphylococcus aureus, Enterococcus faecalis and Bacillus subtilis (Parrot et al., 1990, Can. J. Microbiol. 36:123-130). A preliminary characterization of the antibacterial effect of this strain permitted to hypothesize that at least one mutacin was responsible therefor. However, the mutacin(s) of B-Ny266 has yet to be purified. Furthermore, it remains to be determined whether the wide antibacterial activity associated with strain B-Ny266 is attributable to a single proteinaceous substance or shared by two or more proteinaceous substances therein (Morency et al., 1995, Can. J. Microbiol. 41:826-831; and references therein). It follows, that the determination of the amino sequence of the antibacterial substance(s) present in B-Ny266 had yet to be carried out.
There thus remains a need to purify to homogeneity the bacterial substance(s) carrying the demonstrated antibacterial activity present in S. mutans strain B-Ny266. There further remains a need to identify the amino acid sequence of this substance.
The present invention seeks to meet these and other needs.
The present description refers to a number of documents, the content of which is herein incorporated by reference.
The invention concerns the purification to homogeneity of the proteinaceous substance having bactericidal activity produced by Streptococcus Mutans B-Ny266. More particularly, the present invention concerns the purification to homogeneity of a lantibiotic type A produced by Streptococcus mutans B-Ny266, hereinafter termed mutacin B-Ny266.
The invention also relates to the amino acid sequence of mutacin B-Ny266. More particularly, the invention relates to the primary amino acids sequence of pro-B-Ny266 the precursor protein of mutacin B-Ny266: F-K-S-W-S-F-C-T-P-G-C-A-K-T-G-S-F-N-S-Y-C-C (SEQ ID NO:1). Further, the present invention relates to pro-B-Ny266 sequence being posttranslationnally modified to yield the proposed sequence: F-K-A-W-U-F-A-Abu-P-G-A-A-K-O-G-A-F-N-U-Y-A (SEQ ID NO:4; underlined residues participate in lanthionine or b-methyllanthionine bridges).
The present invention further relates to methods of purifying to homogeneity Mutacin B-Ny266 from S. mutants strain B-Ny266. Further, the invention relates to methods of purifying to homogeneity type A mutacins from S. mutants being close relatives to mutacin B-Ny266. More particularly, the invention relates to methods for the preparation of mutacin B-Ny266 and pharmaceutically acceptable salts thereof comprising cultivating microorganisms capable of expressing mutacin B-Ny266, preferably S. mutants B-Ny266, under condition whereby mutacin B-Ny266 is expressed, isolated, secreted and extracted from cell pellets of the strain expressing B-Ny266 in the culture medium, and if desired, converting the isolated mutacin B-Ny266 to a pharmaceutically acceptable salt thereof, S. Mutans B-Ny266 has been deposited at the American Type Culture Collection (ATCC, 12301 Parklawn Drive, Rockville, Md.) on Aug. 22, 1997 and granted the accession number ATCC 202022.
The invention in addition relates to pharmaceutical compositions comprising mutacin B-Ny266 and pharmaceutically acceptable carriers or excipients. Further, the invention relates to pharmaceutical compositions comprising as active ingredient, mutacin B-Ny266 or a pharmaceutically acceptable salt thereof, and associated with a pharmaceutically acceptable carrier or excipient.
The present invention therefore further relates to a purified preparation of mutacin B-Ny266. More particularly, the invention relates mutacin B-Ny266 purified to more than 80% homogeneity more preferably to more than 90% homogeneity and in a further preferred embodiment to mutacin B-Ny266 purified to approximately 99% homogeneity.
The applicant was the first to purify a Streptococcus mutans mutacin to homogeneity. More particularly, the applicant is the first to purify mutacin B-Ny266 to homogeneity and to determine its complete amino acids sequence. The applicant is therefore the first to obtain a complete amino acids sequence of a lantibiotic produced by S. mutants. 
Before the present invention, it was unclear whether S. mutants strain B-Ny266 antibacterial activity was determined by a single proteinaceous substance. Further, prior to the present invention, in view of the difficulty in purifying to homogeneity mature, modified peptides such as lantibiotic, mutacin B-Ny266 had yet to be obtained in a pure form. The applicant is thus the first to provide antibacterial pharmaceutical compositions comprising mutacin B-Ny266.
The amino acid sequence of mutacin B-Ny266 completely differs from that of streptococcin A-FF22, salivariucin A, and mutacin J-T8. It is however somewhat apparented to the sequence of epidermin and gallidermin although it differs therefrom by five amino acid residues.
It is noteworthy that gallidermin and epidermin only differ from each other by one amino acid while their antibacterial activity have distinguishing features. Contrary to epidermin (Peschel et al., 1996, J. Bacteriol. 178:531-536; Peschel et al., 1997, Mol Gen Genet 254:312-318; Schnell et al., 1988, Nature 333:276-278; Schnell et al., 1992, Eur. J. Biochem. 204:57-68) and like gallidermin (Schnell et al.,1989, FEMS Microbiol. Lett. 58:263-268), the genes coding for mutacin B-Ny266 are located on the chromosome (Mota-Meira et al., 1996, FEMS Symposium on lactic acid bacteria, The Netherlands).
Mutacin B-Ny266 has a molecular mass of 2270 Da while epidermin and gallidermin have a molecular mass of 2164 Da (Nes et al., 1996, Antonie van Leeuwenhoek 69:89-97). Apart from subtilin, it is the only lantibiotic of type A with a net charge of +2 (Nes et al., 1996, Antonie van Leeuwenhoek 69:89-97). It is the only type A lantibiotic with a phenylalanine N-terminal residue (Nes et al., 1996, Antonie van Leeuwenhoek 69:89-97) and it contains 3 phenylalanine residues like cytolysin LS from Enterococcus faecalis (Gillmore et al., 1994, J. Bacteriol. 176:7335-7344) as opposed to none in nisin (Gross et al., 1971, J. Am. Chem. Soc. 93:4634-4635) and two in epidermin/gallidermin.
The above-mentioned features clearly indicate that mutacin B-Ny266 is unique among lantibiotics.
In accordance with the present invention, there is therefore provided, an isolated mutacin B-Ny266 polypeptide comprising the amino acid sequence of pro-B-Ny266: F-K-S-W-S-F-C-T-P-G-C-A-K-T-G-S-F-N-S-Y-C-C (SEQ ID NO:1), or functional derivatives thereof. Additionally, there is also provided an isolated mutacin B-Ny266 polypeptide comprising the amino acid sequence of pro-B-Ny266: F-K-S-W-S-F-C-T-P-G-C-A-K-T-G-S-F-N-S-Y-C-C (SEQ ID NO:1), or functional derivatives thereof, modified into a mature mutacin B-Ny266 polypeptide, comprising amino acid sequence: F-K-A-W-U-F-A-Abu-P-G-A-A-K-O-G-A-F-N-U-Y-A (SEQ ID NO:4), or functional derivatives thereof, wherein underlined residues participate in lanthionine or b-methyllanthionine bridges.
In accordance with the present invention, there is also provided, a substantially pure preparation of mutacin from Streptococcus mutans, having a structure according to FIG. 1.
In accordance with another aspect of the present invention, there is provided, bactericidal and/or bacteriostatic composition comprising an effective amount of the isolated mutacin B-Ny266 polypeptide of the instant invention, and a suitable carrier or excipient. As well there is provided a pharmaceutical composition for the treatment and/or prophylaxis of bacterial strains susceptible to mutacin B-Ny266, comprising an effective amount of the isolated mutacin B-Ny266 polypeptide of the instant invention, and a pharmaceutically acceptable carrier or excipient.
In accordance with yet another aspect of the present invention, there is provided a method of treating a bacterial infection in an animal, comprising the step of administering a therapeutically effective amount of a pharmaceutical composition according with the instant invention.
As well there is provided a Streptococcus mutans strain expressing mutacin B-Ny266 comprising the amino acid sequence of pro-B-Ny266: F-K-S-W-S-F-C-T-P-G-C-A-K-T-G-S-F-N-S-Y-C-C (SEQ ID NO:1), or functional derivatives thereof and a Streptococcus mutans strain expressing a mature mutacin B-Ny266 polypeptide, comprising amino acid sequence: F-K-A-W-U-F-A-Abu-P-G-A-A-K-O-G-A-F-N-U-Y-A (SEQ ID NO:4), or functional derivatives thereof, wherein underlined residues participate in lanthionine or b-methyllanthionine bridges.
In accordance with the present invention, there is additionnally provided a method of producing mutacin B-Ny266 having amino acid sequence: F-K-A-W-U-F-A-Abu-P-G-A-A-K-O-G-A-F-N-U-Y-A (SEQ ID NO:4), or functional derivatives thereof, wherein underlined residues participate in lanthionine or b-methyllanthionine bridges, the method comprising the steps of cultivating a S. Mutans strain expressing the mutacin, in a suitable culture medium enabling expression, production and maturation thereof, and isolating the mutacin B-Ny266. Also there is provided such a method, wherein said mutacin B-Ny266 is isolated by treating the suitable culture medium with a weak hydrophobic adsorber which adsorbs mutacin B-Ny266, and eluting the mutacin B-Ny266 from the adsorber using a suitable organic solvent.
In accordance with the present invention there is provided a method for inhibiting sensitive bacteria comprising: exposing the bacteria to an inhibitory amount of the mutacin B-Ny266 polypeptides of the instant invention.
As used herein, the designation xe2x80x9cfunctional derivativesxe2x80x9d denote a derivative of mutacin B-Ny266 having a related amino acid sequence thereof and retaining mutacin B-Ny266 biological activity. These functional derivatives or equivalents may be natural derivatives of may prepared synthetically. Such derivatives include amino acid sequence having substitutions, deletions, or additions of one or more amino acids, provided that the biological activity of mutacin B-Ny266 is conserved. Generally, the substituting amino acid has chemico-physico properties which are similar to the substituted amino acid. The similar chemico-physico properties include, similarities in charge, bulkiness, hydrophobicity, hydrophylicity and the like.
The term xe2x80x9cfunctional derivativesxe2x80x9d is intended to include xe2x80x9cfragmentsxe2x80x9d, xe2x80x9csegmentsxe2x80x9d, xe2x80x9cvariantsxe2x80x9d, xe2x80x9canalogsxe2x80x9d or xe2x80x9cchemical derivativesxe2x80x9d of the mutacins of the present invention.
As used herein, xe2x80x9cchemical derivativesxe2x80x9d meant to cover additional chemical moieties not normally part of the invention. Such moieties could affect the physico-chemical characteristic of the mutacin derivative (i.e. solubility, absorption, half life and the like, decrease of toxicity). Such moieties are examples of described in Remington""s Pharmaceutical Sciences (1980). Methods of coupling chemical moieties to a polypeptide are well known in the art.
The term xe2x80x9cvariantxe2x80x9d refers herein to a mutacin having a substantially similar structure and biological activity.
It shall also be understood, that since mutacin B-Ny266 possesses its biological activity after maturation of its original primary amino acid structure, that the person of ordinary skill, will be able to obtain a derivative of mutacin B-Ny266 having an initial primary structure which is different from the primary structure of the pro-mutacin B-Ny266 but can mature into an active mutacin having the identical primary structure as that of mutacin B-Ny266.
The functional derivatives of the present invention can be synthesized chemically or produced through recombinant DNA technology. Non-limiting examples of chemical synthesis include solid phase synthesis using for example a polypeptide synthesis apparatus. Recombinant DNA technology methods are also well known in the art. For example, methods of creating fusion proteins by adding additional amino acid, for example at the amino terminal is considered to be within the scope of what is considered a functional variant.
As used herein, the term xe2x80x9cpurifiedxe2x80x9d refers to a molecule having been separated from a cellular component. Thus, a xe2x80x9cpurified proteinxe2x80x9d has been purified to a cell not found in nature. Herein, the designation xe2x80x9csubstantially purexe2x80x9d denotes a preparation that is lacking in all other cellular components. As will be taught herein in a preferred embodiment, the substantially pure preparation of mutacin B-Ny266 is 99.9% pure.
While the method of production of mutacin B-Ny266 of the instant invention is demonstrated by growing Streptococcus mutans strain B-Ny266 in M17 broth, the cultivation of mutacin B-Ny266 producing microorganisms, such as S. mutants can be carried out according to standard methods using conventional culture medium. For instance, S. mutants (ATCC 202022) can be cultivated in a rich medium containing meat extracts, yeast extracts and casein and a carbon source. In one preferred embodiment, lactose is used as the carbon source. In another preferred embodiment, the media used is M17 (BDH). Other media, such as a medium containing a suitable nitrogen source, a carbon source, and a hydroxide of an alkaline earth metal, Ca(OH)2 can also be used. To produce large quantities of the bacterial strain, a suitable bioreactor can be operated. In any event, the methods of growing S. mutans are well known to the person of ordinary skill. It will be understood by the person of ordinary skill that a synthetic media could in some instances provide advantages, since by being less complex, less undesirable components are present in the media, translating in a simpler purification scheme of the mutacin.
Although in a preferred embodiment, mutacin B-Ny266 is purified to homogeneity by a simple method comprising mutacin B-Ny266 extraction from cell pellets. It has also been found that mutacin B-Ny266 may be readily isolated by the simple, expedient method of isolating the mutacin from the culture medium comprising: treating the culture medium with an adsorber followed by a procedure including the steps of adsorption on a weak hydrophobic resin, desorption with methanol or other suitable organic solvents (non-limiting examples thereof includes isopropanol, ethanol, acetonitrile and the like) and further chromatographic purification stages, e.g., reverse phase-HPLC or chromatography on C18 Sep-Pak (Waters) using, e.g., acetonitrile gradient in 0.11% TFA as eluant. In a preferred embodiment, the hydrophobic resin XAD-7 was used in accordance with the method of purification of the present invention. It will be understood by the person of ordinary skill that other types of hydrophobic chromatography columns can be used in the context of the purification of the present invention. Other hydrophobic columns such as XAD-2, XAD-4, XAD-8, XAD-11, XAD-16, XAD-1180, XAD-2000 could also be used, although XAD-7 has been consistently the most efficacious hydrophobic column used.
As the mutacin B-Ny266 is a strongly basic compound, it will readily form salts by addition of suitable acids, including pharmaceutically acceptable acids according to conventional methods. Physiologically acceptable organic or inorganic acids which can be used for this purpose are, for example, hydrochloric acid, hydrobromic acid, sulphuric acid, benzosulphonic acid, methanesulphonic acid, p-toluenesulphonic acid and cyclohexylsulphamic acid.
In view of its spectrum of antibacterial activity, mutacin B-Ny266 and its acid addition salts are useful for combatting bacteria and for treating bacterial infections. In particular, in view of their activity against important strains of Actinobacilli, Bacillus sp., Clostridium sp., Corynebacteria, Enterococci, Listeria monocytogenes, Mycobacterium phlei, Neisseria, Propionobactere, Staphylococci, and Streptococci, mutacin B-Ny266 and its acid addition salts are particularly useful in combatting infections caused by Gram-positives bacteria.
Thus according to a further feature of the present invention there are provided pharmaceutical compositions containing, as active ingredient, mutacin B-Ny266 or a pharmaceutically acceptable acid addition salt thereof, in association with one or more inert pharmaceutical carriers and/or excipients. Treatment and prophylaxy of Gram-positive bacterial infections are contemplated as being within the scope of the present invention.
The term pharmaceutical composition as used herein is meant to cover the veterinary field since the mutacin of the invention also finds uses in animal treatment and prophylaxy. Treatment of animals such as cow (mastitis), chicken and the like are within the scope of the present invention.
For pharmaceutical administration, the said polypeptide may be incorporated into preparations in either liquid or solid forms using carriers and excipients conventionally employed in the pharmaceutical art, optionally in combination with further active ingredients. The preparation may, for example, be applied orally, parenterally, enterally or preferably topically. Preferred forms include, for example, solutions, emulsions, gels, sprays, lotions, ointments, creams or powders.
Dosage in pharmaceutical compositions can vary from 0.001 mg/kg to 50 mg/kg of mutacin B-Ny266, in one or more administrations daily, for one or several days. Mutacin B-Ny266 can be administered parenterally by injection or by gradual perfusion over time. It can be administered intravenously, intraperitoneally, intramuscularly, or subcutaneously.
One of ordinary skill can readily determine the amounts of mutacin to be administered. It is apparent that the dosage will be dependent on the particular treatment used. It should also be clear that the dosage should be chosen to display the biological activity without causing adverse effects. It will be understood that age, sex, type of disease, of formulation and other variables known to the person of ordinary skill will affect determination of the dosage to be used.
The pharmaceutically acceptable carriers and excipients are well known in the art. A representative textbook thereon is Remington""s Paharamaceutical Sciences, 1980, 16th Ed., Mack Eds.
Advantageously the compositions may be formulated as dosage units, each unit being adapted to supply a fixed dose of active ingredient. The total daily dose may, of course, be varied depending on the subject treated and the particular use of the composition. This can obviously be adapted by the treating professional.
If under certain conditions, it was forseen that it would be beneficial to provide a patient with a longer circulating time and/or slow release of mutacin B-Ny266. Under such conditions, the mutacin B-Ny266 can be trapped in well known delivery molecules such as liposomes, synthetic vesicles, nanoerythrosomes (U.S. Pat. No. 5,653,999) and the like, according to known methods.
Mutacin B-Ny266 and its acid addition salts may also be used as an additive in cosmetic formulations especially those containing collagen in which it would act as a biopreservative and/or stabilizer.
A cosmetic preparation according to the present invention will include mutacin B-Ny266 or an acid addition salt thereof in association with a suitable carrier and/or excipient preferably collagen and optionally other additives suitable for cosmetic preparations such as perfumes and coloring agents. Mutacin B-Ny266 can also be added to an oral composition (i.e. toothpaste or mouthwash) in such types of preparations or compositions mutacin B-Ny266 can be added in an amount of 0.0001 to 10 wt %, preferably 0.001 to 5 wt % and more advandtageously 0.01 to 2 wt %.
In foodstuff protecting compositions in which mutacin B-Ny266 prevents solid or liquid food spoiling (i.e. meats, dowry products, beer, wine and the like) inhibiting or billing bacteria and especially harmful bacteria, mutacin B-Ny266 can be added to the composition in the range of 1.25 mg/Kg to 6.25 mg/Kg ot treated food or 1.25 mg/L to 2.5 mg/L of treated food.
Furthermore, mutacin B-Ny266 or an acid addition salt thereof in association with a suitable carrier and/or excipient can be used as biopreservative agent in foods and in personal hygiene products. Once again, the formulation can be adapted by the person of ordinary skill to suit particular needs. In view of the wide spectrum of bactericidal activity of mutacin B-Ny266, mutacin B-Ny266 or an acid addition salt thereof in association with a suitable carrier and/or excipient could further find uses as anticarries agent (i.e. in toothpaste, mouth wash, and in topical application), disinfectant cleanser (to combat acne for example), selective agent against Gram-positive bacteria in culture media (Ray, 1992, in: Food Biopreservative of Microbial Origin, Ray et al., Eds, CRC Press Inc., Boca Raton Ill.:207-264; Harlanda, 1993, in: Bacteriocins of Lactic Acid Bacteria, Hoover et al., Eds., Acad. Press Inc., San Diego, p. 63-91; Weder et al., Canadian Application 1, 277, 617; and Jung et al., U.S. Pat. No. 5,231,013).